PROJECT SUMMARY Breast cancer is one of the most diagnosed type of cancer and second leading cause of cancer-related death among women in the U.S. Treatment remains challenging due to the heterogeneity and highly dynamic nature of breast tumors. Although early detection through screening has largely contributed to a decline in breast cancer-related mortality, tumor recurrence and metastasis still remain associated with poor prognosis. Once the disease becomes metastatic, the median survival is around 12 to 15 months. But more alarming is the incidence of the triple-negative subtype, the most aggressive type of breast cancer, in the younger population. Increasing evidence identifies the epithelial-mesenchymal transition process as a potential contributor of metastasis in this subtype. In fact, breast carcinoma cells that undergo EMT acquire invasive properties that contribute to the formation of metastases. In addition, EMT results in increased expression of extracellular matrix proteins including fibronectin that enhance tumor invasion, regulate cancer cell proliferation, and limit tumor cell responsiveness to therapies. Therefore, there is an urgent need to identify novel molecular targets in breast cancer-associated EMT, and develop new therapeutics to prevent triple-negative breast cancer progression through metastasis. In our previous published research we observed a loss of connexin43 (Cx43) gap junctions at the plasma membrane concomitantly with an increase in intracellular Cx43 expression during EMT. Despite several studies in the field, the role of Cx43 in breast cancer remains ambiguous with roles in suppressing tumor growth as well as facilitating tumor progression and metastasis. Our preliminary research identifies a tumorigenic role for Cx43 in EMT and triple-negative cancer cells through its interaction with microtubules. Regulating localization and activity of Cx43 is associated with the multiple sites for protein?protein interaction within the Cx43 carboxy-terminus (CT). The Cx43 CT includes a tubulin binding domain and we developed a novel Cx43 mimetic peptide named JM2 (juxtamembrane 2) composed of the Cx43 CT encompassing the microtubule binding sequence. Our goal is to assess the therapeutic effect of JM2 to target triple-negative breast cancer in cell-based assays in vitro and in metastatic breast cancer mouse models in vivo. Our proposed research aims to validate Cx43 as a novel molecular target in triple-negative breast cancer, and use JM2 to disrupt Cx43- microtubule interaction without affecting Cx43 expression, thus regulating Cx43 localization and activity more specifically. This proposal aims to fill a gap in knowledge, whereby no previous studies evaluated the tumorigenic role of Cx43 through interaction with microtubules in breast cancer cells and no efficient targeted therapeutics currently exist that can successfully ablate the metastatic potential of triple-negative breast cancer cells.